Two-photon live imaging of direct glia-to-neuron conversion in the mouse cortex

Over the past decade,a growing number of studies have reported transcription factor-based in situ reprogramming that can directly conve rt endogenous glial cells into functional neurons as an alternative approach for n euro regeneration in the adult mammalian central ne rvous system.Howeve r,many questions remain regarding how a terminally differentiated glial cell can transform into a delicate neuron that forms part of the intricate brain circuitry.In addition,concerns have recently been raised around the absence of astrocyte-to-neuron conversion in astrocytic lineage-tra cing mice.In this study,we employed repetitive two-photon imaging to continuously capture the in situ astrocyte-to-neuron conversion process following ecto pic expression of the neural transcription factor NeuroD1 in both prolife rating reactive astrocytes and lineage-tra ced astrocytes in the mouse cortex.Time-lapse imaging over several wee ks revealed the ste p-by-step transition from a typical astrocyte with numero us short,tapered branches to a typical neuro n with a few long neurites and dynamic growth cones that actively explored the local environment.In addition,these lineage-converting cells were able to migrate ra dially or to ngentially to relocate to suitable positions.Furthermore,two-photon Ca2+imaging and patch-clamp recordings confirmed that the newly generated neuro ns exhibited synchronous calcium signals,repetitive action potentials,and spontaneous synaptic responses,suggesting that they had made functional synaptic connections within local neural circuits.In conclusion,we directly visualized the step-by-step lineage conversion process from astrocytes to functional neurons in vivo and unambiguously demonstrated that adult mammalian brains are highly plastic with respect to their potential for neuro regeneration and neural circuit reconstruction.

Loss of CO_2 sensing by the olfactory system of CNGA3 knockout mice

Atmospheric CO2 can signal the presence of food, predators or environmental stress and trigger stereotypical behaviors in both vertebrates and invertebrates. Recent studies have shown that the necklace olfactory system in mice sensitively detects CO2 in the air. Olfactory CO2 neurons are believed to rely on cyclic gnanosine monophosphate （cGMP） as the key second messenger; however, the specific ion channel underlying CO2 responses remains unclear. Here we show that CO2-evoked neuronal and behavioral responses require cyclic nucleotide-gated （CNG） channels consisting of the CNGA3 subunit. Through Ca2＋-imaging, we found that CO2-triggered Ca2＋ influx was abolished in necklace olfactory sensory neurons （OSNs） of CNGA3-knockout mice. Olfactory detection tests using a Go/No-go paradigm showed that these knockout mice failed to detect 0.5% CO2. Thus, sensitive detection of atmospheric CO2 depends on the function of CNG channels consisting of the CNGA3 subunit in necklace OSNs. These data support the important role of the necklace olfactory system in CO2 sensing and extend our understanding of the signal transduction pathway mediating CO2 detection in mammals [Current Zoology 56 （6）： 793-799, 2010].

Biophotons Contribute to Retinal Dark Noise

The discovery of dark noise in retinal photoreceptors resulted in a long-lasting controversy over its origin and the underlying mechanisms.Here,we used a novel ultra-weak biophoton imaging system（UBIS） to detect biophotonic activity（emission） under dark conditions in rat and bullfrog（Rana catesbeiana） retinas in vitro.We found a significant temperature-dependent increase in biophotonic activity that was completely blocked either by removing intracellular and extracellular Ca^（2＋）together or inhibiting phosphodiesterase 6.These findings suggest that the photon-like component of discrete dark noise may not be caused by a direct contribution of the thermal activation of rhodopsin,but rather by an indirect thermal induction of biophotonic activity,which then activates the retinal chromophore of rhodopsin.Therefore,this study suggests a possible solution regarding the thermal activation energy barrier for discrete dark noise,which has been debated for almost half a century.